A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. At least 10% of pathogenic mutations in membrane proteins (MPs) result in mis- orientation of their transmembrane domains (TMDs). Most studies focus on the protein and the membrane insertion machinery with little consideration of how lipid environment affects TMD organization. The long-term goal of this proposal is to understand the role of lipid-protein interactions in the assembly, structure and function of MPs. Using a combined molecular genetic and biochemical approach, we established that lipid- dependent TMD orientation is dynamic during and after MP assembly in vivo and is independent of other cellular factors in vitro. Dependence of TMD topology solely on the intrinsic properties of a MP and its lipid environment indicates a thermodynamically driven process that can occur in any cell membrane at any time. We developed a set of lipid mutants of Escherichia coli in which lipid composition can be regulated during or temporally after MP insertion. This mutant set and a new proteoliposome system in which lipid composition can be controlled before and after MP reconstitution will be used to test kinetic and thermodynamic limits of TMD interconversions and establish direct lipid-protein interactions as the basis for in vivo observed phenotypes. Combining studies of MP and lipid properties led to our formulation of the Charge Balance Rule, which postulates that charge interactions between lipid head groups and MP extramembrane domains (EMDs) are a determinant of MP topology. This rule explains stable, dynamic and dual topological organization of a MP and provides a proof of principle for lipid-dependent assembly of MPs in more complex eukaryotic systems. In Aim1 we will determine the limits imposed on lipid-induced post-assembly changes in MP topology by post- translation glycosylation and phosphorylation of EMDs. We will determine whether the rate of phosphorylation- triggered topological changes occur rapidly enough to represent a novel mechanism for metabolic regulation linked to MP dynamic organization. We will extend our studies to FtsK whose phosphorylation appears to alter TMD topology as a regulatory mechanism during cell division of E. coli. In Aim 2 we will build biologically based membrane insertion scales for individual amino acids that incorporate lipid composition, which has not been previously considered. Understanding how lipid composition affects TMD insertion and orientation will provide a molecular basis for initial TMD orientation, reversible post-assembly TMD reorientation, MP topological heterogeneity, and misfolding of mutant or heterologously expressed MPs. In Aim 3 we will analyze how lipid composition, lipid transbilayer asymmetry and membrane bioenergetic parameters modulate the effective net charge of EMDs, which will provide an understanding at the mechanistic level of the Charge Balance Rule. Extrapolating new MP assembly rules to eukaryotic cells will provide insight into the molecular basis for diseases resulting from misfolded MPs.